Background: Ischemic stroke is one of the leading causes of morbidity, mortality, and disability worldwide [1]. Human neural progenitor cells (hNPCs) therapy offers a novel means for treating ischemic stroke, and has shown great promise in clinical translation [3,4]. However, the dynamic cell fate after hNPCs transplantation remain poorly understood. Positron emission tomography (PET) enables the visualization, characterization, and quantification of biologic processes at cellular and molecular levels in vivo. ¹⁸F-SynVesT-1 is a novel radiopharmaceutical for measuring synaptic density in vivo [5], enabling the evaluation of dynamic synaptic repair after hNPCs transplantation post-stroke. Besides, single-nucleus sequencing (snRNA-seq) is a rapidly evolving technology used to analyze gene expression profiling of single cells on a large-scale. By revealing cell fate of hNPCs in the ischemia-injured cortex, characterizing cellular heterogeneity and complex interactions, snRNA-seq is able to provide insights into disease pathophysiology, and facilitate the development of novel therapies [6,7].

Objective: This study aimed to explore the dynamic synaptic repair of ischemic stroke after hNPCs transplantation, and created the first snRNA-seq atlas of hNPCs cell fate within the ischemic stroke-damaged cortex.

Methods: Sprague-Dawley rats were subjected to photothrombotic cerebral infarction. Intracortical transplantation of hNPCs was performed stereotaxically 2 weeks in peri-infarct areas. Bioluminescence imaging (BLI) was performed to assess survival and proliferation of hNPCs. ¹⁸F-SynVesT-1 and ¹⁸F-FDG PET/CT imaging were used to evaluate synaptic density and cerebral glucose metabolism. Post-mortem histological analysis including immunofluorescent staining and immunoelectron microscopy were used to assess hNPCs differentiation and synaptic connections. Whole-cell patch-clamp recordings were used to assess synaptic function of hNPCs. SnRNA-seq was performed on the grafted area at 2-, 4- and 12-weeks post-transplantation. snRNA-seq was used to characterize the cell fate and molecular signals of graft at the single-nucleus scale.

Results: BLI demonstrated the long-term survival of hNPCs in the infracted brain. ¹⁸F-SynVesT-1 PET imaging revealed dynamic synaptic changes after hNPC transplantation in ischemic stroke. Immunofluorescent staining results indicated that hNPCs predominantly differentiated into various neuronal cells, expressing cortical layer-specific markers. Immunoelectron microscopy identified synaptic connections between the grafts and host neurons. Additionally, hNPCs progeny cells extended axons into the infracted area and contralateral corpus callosum. Whole-cell patch-clamp recordings confirmed that hNPCs progeny cells exhibited electrophysiological properties and established effective connections with surrounding cells. SnRNA-seq revealed the composition of human-derived cells, including intermediate progenitor cells, radial glia cells, astrocytes, and excitatory neurons. hNPCs differentiated into neurons, originating from distinct lineages. Cell-cell communication analysis revealed the important role of radial glial cells in early phase. Synapse-related genes among neural cells presented different expression patterns, which might serve as potential targets for promoting synaptogenesis, and enhancing the efficacy of stem cell therapy.

Conclusions: Grafted hNPCs proliferated, migrated, and differentiated into various neuronal cell types within the ischemic stroke-damaged cortex. The grafts established synaptic connections and communication with surrounding cells. ¹⁸F-SynVesT-1 PET is a non-invasive imaging biomarker for quantifying the dynamic synaptic changes after hNPCs transplantation in ischemic stroke. A snRNA-seq atlas of hNPCs fate in the ischemia-injured cortex was established, and offered potential interventional targets for further enhancing the efficacy of stem cell therapy.

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Image/Figure Caption:

(a) Representative T2-weighted MRI and TTC staining of stroke-injured areas at 2 days after photothrombotic cerebral infarction.

(b) The BLI signals in the ischemic brain tissues at weeks 2, 4, 8 and 12 after cell transplantation, n = 8.

(c) Representative [¹⁸F]SynVesT-1 PET image and quantification of SUVR in the control (n=5) and hNPCs transplantation group (n=7) at weeks 2, 4 and 12 after cell transplantation.

(d) Representative immunostaining of cortical layer-specific markers CTIP2, TBR1, BRN2, and SATB2. Quantification of the percentages of CTIP2, TBR1, BRN2, and SATB2 positive cells in human derived cells at weeks 2, 4 and 12 after cell transplantation. Scale bar = 50 μm.

(e) Low magnification (Scale bar = 1 mm) and high magnification (Scale bar = 50 μm) illustrated the axonal projection pathways and territory of transplanted neurons. The red arrowheads indicated GFP+ fibers in the infracted area and corpus callosum.

(f) Grafted human hNPC-derived neurons expressed synaptophysin (SYP) (Scale bar = 10 μm) and established synapses with host neurons. Whole-cell patch-clamp recordings showed action potentials and postsynaptic currents of grafted cells.

(g) Sn-RNA seq tissues were acquired from red frame. Identification of hNPC-derived cell types and percentages. Pseudotime analysis indicated hNPC-derived cell development trajectories

(h) hNPC-derived neurons were divided into eight subtypes. Quantification of the percentage of neuron subtypes at different developmental stages. Pseudotime analysis indicated hNPC-derived neuron development trajectories

(i) Heatmap of pathways enriched for each neuron subtype-specific genes by GO analysis.

(j) Heatmap of the expression level of top 10 genes in the eight hNPC-derived neuron subtypes.

(k) Violin plots of the expression level of trans-synaptic cell-adhesion molecules (CAMs) related genes in the hNPC-derived neuron.

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